The oxygen carrying portion of the red blood cell is hemoglobin, a tetrameric protein molecule composed of two identical alpha globins (.alpha..sub.1, .alpha..sub.2), two identical beta globins (.beta..sub.1, .beta..sub.2) and four heme molecules. A heme molecule is incorporated into each of the alpha and beta globins to give alpha and beta subunits. Heme is a macrocyclic organic molecule that contains an iron atom at its center; each heme can combine reversibly with one ligand molecule, for example oxygen. In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form two stable alpha/beta dimers, which in turn associate to form the tetramer (a homodimer). The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges.
In the unliganded state (deoxygenated or "deoxy") state, the four subunits form a quaternary structure known as "T" (for "tense") state. During ligand binding, the .alpha..sub.1 .beta..sub.1 and .alpha..sub.2 .beta..sub.2 interfaces remain relatively fixed while the .alpha..sub.1 .beta..sub.2 and .alpha..sub.2 .beta..sub.1 interfaces exhibit considerable movement. When a ligand is bound to the hemoglobin molecule, the globins move with respect to each other, and as a result intersubunit distances are increased relative to the deoxygenated distances. Thus, when a ligand is bound at the heme groups, the molecule assumes the "relaxed" or "R" quaternary structure, the thermodynamically stable form of the molecule when ligands are bound at three or more hemes.
Ligands, particularly oxygen, bind reversibly to the reduced form of the iron (ferrous, Fe.sup.+2) in the heme. When the iron in the heme is oxidized to Fe.sup.+3 (the ferric form of iron), oxygen and some other ligands cannot bind to the iron of the heme and the hemoglobin is non-functional in terms of oxygen transport. The iron in the heme groups may be oxidized in a number of fashions. For example, the iron may oxidize through a pathway mediated by binding of a water molecule at the heme iron to produce methemoglobin ("autoxidation"). Autoxidation can be enhanced by the presence of trace metals in solution. Methemoglobin can also be produced as a result of direct oxidation by chemicals with higher redox potentials, such as ferricyanide, or by indirect oxidation by reducing agents through a hydrogen peroxide mediated pathway (Castro, C. E. et al., in: Biochemical and Clinical Aspects of Hemoglobin Abnormalities, Academic Press, Inc., pp 495-503, 1978). Furthermore, deoxyhemoglobin can be oxidized to methemoglobin by chemicals such as ferricyanide even in the absence of oxygen. The protein itself may be oxidized as well, without concommittant oxidation of the iron in the heme groups. For example, hemoglobin may be oxidatively denatured by chemicals such as hydrazine without going through a methemoglobin intermediate. (see Bunn, H. F. and Forget, B. G. Hemoglobin: Molecular, Genetic and Clinical Aspects, W.B. Saunders Company, Philadelphia, pp 634-662 for review).
However produced, methemoglobin is a non-functional form of hemoglobin that cannot bind oxygen or carbon monoxide and shows significantly altered nitric oxide binding characteristics. Methemoglobin molecules are vulnerable to accelerated degradation due to hemichrome formation, heme loss, precipitation, reaction with hydrogen peroxide to form toxic radicals and the like.
In addition to the reduction of functionality of a hemoglobin solution by the formation of methemoglobin, the protein portions of the hemoglobin molecule can be modified and altered by oxidative damage. For example, the oxidation of the iron of the heme results in the production of hydrogen peroxide (Watkins, J. A. Bioc. Biophys. Res. Comm. 132: 742-748, 1985) as well as superoxide (Thillet, J. and Michelson, A. M., Free Rad. Res. Comm. 1: 89-100, 1985). These activated oxygen species can then damage the hemoglobin protein, for example, by causing polymerization of the molecule (Thillet, J., supra) or by damaging individual amino acids and thereby disrupting tertiary structure (Stefek, R. P. and Thomas, M. J., Free Rad. Res. Comms. 12-13: 489-497, 1991). These changes can result in increased immunogenicity (Riechlin, M., Adv. Immunol. 20: 71-132, 1975; Noble, R. W. et al. Bioc. 11: 3326-3332, 1972). Ultimately, damage resulting from activated oxygen species, irrespective of source, may lead to oxidative denaturation of the molecule and its precipitation. Therefore, during storage of hemoglobin solutions, the avoidance of oxidation, whether of the protein itself or of the heme groups within the protein, is necessary to maintain functionality and the tertiary and quaternary structure of hemoglobin in solution and to limit immunogenicity.
It has long been known that hemoglobin solutions form methemoglobin more slowly if stored under deoxygenated conditions rather than oxygenated conditions (Antonini and Brunori, Hemoglobin and Myoglobin in Their Reactions with Ligands, North Holland Publishing Company, Amsterdam 13-39, 1971; Di lorio, E. E., Meth. Enzymol. 76: 57-72, 1981). However, preservation of hemoglobin under deoxygenated conditions poses significant technical difficulties (Di Iorio, E. E., Meth. Enzymol. 76: 57-72, 1981), or requires the addition of potentially toxic chemicals. Moreover, the choice of exogenous chemical agents has been extremely difficult since these additives can act as oxidants or reductants, depending on the conditions of the solution and unpredictable protein/agent interactions (Akers, M. J. J. Parent. Sci. Tech. 36: 222-228, 1982). In the case of hemoglobin solutions, the choice of an exogenous chemical agent that will act as a reductant is further complicated by the fact that not all reducing agents can reduce hemoglobin, and some reducing agents may act as oxidants in a given hemoglobin formulation (Eyer, P., Mol. Pharmacol. 11: 326-334, 1975; Kikugawa, K. Chem. Pharm. Bulletin 29: 1382-1389, 1981; Stratton, L. P. Hemoglobin 12: 353-368, 1988). If the exogenous chemical does act to reduce methemoglobin formation in solution, there may be unexpected side reactions that can affect the protein structure (Antonini and Brunori, Hemoglobin and Myoglobin in Their Reactions with Ligands, North Holland Publishing Company, Amsterdam 13-39,1971).
In order to reduce or eliminate the need for exogenous reducing agents during storage, Nho (PCT publication WO 92/08478) designed an apparatus which allowed the rapid deoxygenation of hemoglobin solutions. Hemoglobin solutions were deoxygenated by passing the solution through one side of a gas exchange device and passing an inert gas, nitrogen, on the other side of a gas permeable membrane. The hemoglobin was circulated until it was at least 90% deoxygenated. No exogenous reductants were added to the hemoglobin solution; oxidation, although not completely eliminated, was slowed by careful removal of oxygen in the hemoglobin solution. Thus there was no introduction of chemical reductants which might elicit unforeseen biological responses when used in pharmaceutical formulations, or which might react with the hemoglobin itself in an unpredictable fashion.
A different approach to the storage of hemoglobin solutions was described by Kandler, R. L. and J. C. Spicuzza (U.S. Pat. No. 5,352,773 and PCT publication WO 92/02239). They utilized the intrinsic ability of a purified di-aspirin crosslinked hemoglobin solution to "auto-reduce" in the absence of any exogenous chemical reductants. In their solutions, methemoglobin concentrations were reduced by storing the solutions in overwrapped, oxygen impermeable containers. Some of these overwrapped containers contained oxygen scavenging pouches placed between the overwrap and the inner container. Methemoglobin concentrations as high as 50% could be reduced to levels as low as 1.5% by storage alone as long as oxygen was rigorously excluded from the containers during the storage of the solution. This methodology did not depend on whether or not the initial solution was deoxygenated prior to storage. However, even though this methodology did not require the addition of exogenous reducing agents, the material was held for long periods of time to ensure methemoglobin was reduced to clinically acceptable levels, and/or it was held at relatively high temperatures. Extended storage or storage at elevated temperatures might result in modifications of the hemoglobin structure or growth of microorganisms if inadequately sterilized.
Deoxygenation of hemoglobin solutions alone may not provide sufficient stability to the hemoglobin solutions to allow for long term storage, especially storage at room temperature. For example, DeVenuto (DeVenuto, F., J. Lab. Clin. Med. 92: 946-952, 1978) found that deoxygenated hemoglobin solutions showed more rapid methemoglobin formation than comparable solutions stored in the presence of oxygen. Moreover, he was not able to demonstrate any solution that showed stability at room temperature.
Ascorbate (ascorbic acid or vitamin C) has been commonly used both in pharmaceutical compositions and as a reagent to scavenge oxygen or act as an antioxidant in hemoglobin compositions. As a pharmaceutical, ascorbate has been administered directly to treat methemoglobinemia in vivo (Kiese, M. Methemoglobinemia: A Comprehensive Treatise, CRC Press, Inc., Cleveland, Ohio, pg. 23, 1974; Deeny, J., et al., Br. Med. J. 1: 721-723, 1943). Ascorbate has been used as a preservative and a stabilizer in many protein solutions, particularly proteins derived from blood products. For example, G. P. Wiesehahn et al. (U.S. Pat. No. 4,727,027) described the decontamination of solutions of biologically active proteins derived from blood or blood components, particularly Factor VIII, by photodecontamination. They stabilized solutions of Factor VIII prior to photodecontamination by deoxygenating the solutions either by the addition of high concentrations of oxygen scavengers, such as 10 mM ascorbate, by flushing with inert gases, or both addition of oxygen scavengers and flushing with inert gas. Osterber et al. (PCT publication WO 94/26286) also describe a stabilized, deoxygenated formulation of factor VIII augmented with antioxidants such as glutathione, acetylcysteine, methionine, tocopherol, butyl hydroxy toluene, butyl hydroxy anisole or phenolic compounds. Although these antioxidants are suitable for use at low doses and are thus appropriate for the small dosage volumes of Factor VIII, many of these antioxidants can be toxic at high doses and hence are not appropriate for use in formulations where high volumes of a therapeutic, for example, hemoglobin, might be administered. In addition, the application is silent with respect to the interaction of the antioxidants and hemoglobin. It is of note that Osterber et al. report that the addition of ascorbate to a deoxygenated solution of Factor VIII resulted in reduced Factor VIII stability.
As early as the 1940's, high concentrations of ascorbate were used to reduce methemoglobin in hemoglobin compositions (Gibson, Q. H., Bioc. I. 37: 615-618, 1943). However, the interaction of ascorbate and hemoglobin have been unpredictable. For example, Kikugawa et al. (Kikugawa, K. et al., Chem. Pharm. Bull. 29: 1382-1389, 1981) noted that ascorbate acted as a prooxidant, in fact enhancing oxidation, when added to oxygenated formulations of hemoglobin. Kramlova et al. (Kramlova, M. et al., Haematologia 10: 365-371, 1976 ) and Stratton et al., supra, suggested that addition of ascorbate to hemoglobin solutions also resulted in enhanced oxidation rather than protection from oxidation.
Ascorbate at relatively high concentrations has been used with some success to stabilize deoxygenated hemoglobin solutions. For example Bonhard et al. (U.S. Pat. No. 4,777,244) used ascorbate as an oxygen scavenger in a hemoglobin solution prior to crosslinking deoxygenated hemoglobin. They emphasized the need to use high levels of ascorbate (at least 4 moles of ascorbic acid per mole of hemoglobin) to ensure that all oxygen was scavenged in the hemoglobin solution. Kothe et al. (Kothe, N., Eichentopf, B. and Bonhard, K. Surg. Gyn. Obst. 161: 563-569 1985) also used ascorbate to stabilize a deoxygenated hemoglobin solution. These authors used 5.45 mM ascorbate in a 8.5 gm/dl hemoglobin solution (.about.4 moles ascorbate per mole of hemoglobin) and reported no significant formation of methemoglobin in the hemoglobin formulation after storage at 4.degree. C. for one year. Clerc et al. (Clerc, Y. et al., Service de Sante des Armees Trav. Scient. 8: 211, 1987) reported similar low levels of methemoglobin formation in a formulation based on the Kothe solution (4:1 molar ratio ascorbate to hemoglobin) during storage for 11 months at 4.degree. C. Kikugawa et al., supra, reported that dilute (250 mg/dl) deoxygenated hemoglobin solutions treated with 5 mM ascorbate (.about.13:1 molar ratio ascorbate to hemoglobin) were stable for the 60 minutes of their study. Long term stability of deoxygenated ascorbate containing solutions was not discussed by these authors. Nho et al. (U.S. Pat. No. 5,234,903) followed methemoglobin formation in a 5-6 gm/dl deoxygenated bovine hemoglobin solution and found that 30 mM cysteine was 5 times more effective for the prevention of methemoglobin formation than the same amount of ascorbate (.about.35:1 molar ratio ascorbate:hemoglobin). Moreover, the addition of ascorbate to their hemoglobin formulation resulted in significant release of free iron. Indeed, Estep discussed the requirement that reductants that are added to hemoglobin solutions must have redox potentials higher than ascorbate to maintain these solutions under deoxygenated conditions; ascorbate is simply not sufficiently reducing to maintain the deoxygenation of hemoglobin solutions (Estep, T. N. U.S. Pat. No., 4,861,867). Note that all compositions that have used ascorbate as an antioxidant or an oxygen scavenger have used at least a 4:1 molar ratio of ascorbate to hemoglobin.
Storage of previously known hemoglobin formulations at room temperature has proven problematic (Keipert, P. E. and Chang, T. M. S., Biomat., Art. Cells, Art. Org., 16: 185-196, 1988; Christensen, S. M. et al., J. Bioc. Biophys. Meth. 17: 143-154, 1988; Moore, G. L. et al., Artif. Organs 16: 513-518, 1992). Recently, however, Kandler and Spicussa (PCT publication WO 92/02239) and Nho (PCT publication WO 92/08478) have been able to demonstrate slow or no methemoglobin formation in hemoglobin solutions stored under deoxygenated conditions without addition of exogenous chemical reductants at room temperature for periods of up to 10 months.
The present invention is based on the surprising finding that hemoglobin undergoes significant modifications that may have physiological implications even prior to the appearance of significant quantities of methemoglobin. Furthermore, it has been discovered that there is a relationship between the amount of oxygen in a hemoglobin formulation and the amount of ascorbate required to stabilize the hemoglobin. In addition, it has been surprisingly discovered that certain ratios of hemoglobin, reducing agent and oxygen improve the stability of hemoglobin during storage.